(1-hydroxyethylidene)bisphosphonic acid, potassium salt

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

There are no reproductive toxicity data for HEDP-xK, therefore good quality data are read-across from the category member HEDP (2-3Na).

In the key two-generation feeding study (reliability score 2), conducted to a protocol similar to OECD Test Guideline 416 and pre-GLP, no treatment-related malformations or developmental variations were noted at any tested dose of HEDP (2-3Na). It was concluded that a NOAEL for maternal dams and for the F1 and F2 generations are 447 mg/kg bw/day which is equivalent to 368 mg/kg bw/day active acid (Procter & Gamble, 1971a).

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

Use of 2 dose levels; Sexual milestones in pups/analytical confirmation of dose not given; No sperm analyses (but conception rate of 90% suggests unaffected spermatogenesis, and OECD 416 allows omission if information is available from e.g. 90 day study)

GLP compliance:

no

Remarks:

prior to GLP

Limit test:

no

Species:

rat

Strain:

other: Charles-River

Sex:

male/female

Details on test animals or test system and environmental conditions:

Weanling Charles-River rats were distributed into 5 groups, each composed of 22 females and 22 males, according to body weight and litter. They were housed in stainless steel cages with food (Ground Purina Laboratory Chow) and fresh water furnished ad libitum. Room temperature was maintained at 23 +/- 1°C, and relative humidity at 50 +/- 5%. Lighting was on a 12 hour cycle and background music was employed to equalize ambient noises.
During the first 8 weeks, the rats were caged individually and feed consumption and body weights were recorded at weekly intervals. The rats were then paired and placed into mating cages. This procedure was repeated for the breeders in the second generation (F1b).
Pregnant females were placed in nesting cages which were furnished with a plywood liner and shredded paper.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

When appropriate, HEDP-2Na was mixed with the diet at levels of 0.5 and 0.1%. Both sexes were fed with the respective diets either continuously or only during the time when the females were in their sixth through fifteenth days of gestation.
Females have been determined to be in pro-estrus by vaginal smearing and continuous exposure started "from weaning", i.e. in week 5.

Details on mating procedure:

After 8 weeks, the rats were paired and placed into mating cages. This procedure was repeated for the breeders in the second generation (F1b).

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Exposure period: continuous

Frequency of treatment:

daily

Details on study schedule:

When appropriate, HEDP-2Na was mixed with the diet at levels of 0.5 and 0.1%. Both sexes were fed with the respective diets either continuously or only during the time when the females were in their sixth through fifteenth days of gestation.
During the first 8 weeks, the rats were caged individually and feed consumption and body weights were recorded at weekly intervals. The rats were then paired and placed into mating cages. This procedure was repeated for the breeders in the second generation (F1b).

Pregnancies were timed by vaginal smearing (Long and Evans, 1922), and identification of sperm in the smear designated day 0. The pregnant females were then placed in nesting cages which were furnished with a plywood liner and shredded paper.

The original females (F0) were allowed to deliver their first 2 litters, while the third litters were used for teratologic evaluation. The newborns were counted at birth, but not handled until they were 4 days old. They were then weighed, sexed and inspected grossly. All litters containing more than 8 pups were reduced to that number to equalize the stress on the mothers during the lactation period. After the weaning, weights were recorded, all F1a pups were discarded, but 25 weanlings of each sex, from each treatment group, were selected from the F1b litters for second generation breeding stock.

Twenty (20) pairs of rats from each group were bred and the remaining 5 pairs were necropsied and examined histologically. Subsequently, the first litters of the second generation (F2a) were evaluated similarly to the F1a pups, while the second litters (F2b) were used for teratologic evaluation.
During the teratological phases one-half of each treatment group, selected prior to mating, were sacrificed by excessive ether-inhalation on day 13 and the other half on day 21 of the gestation period.

The pregnant females were examined for number of resorptions, corpora lutea and implantations. The foetuses were dried of amniotic fluid, sexed, carefully inspected for gross abnormalities and weighed. One-third of the foetuses were cleared, stained with Alizarin red stain (Staples and Schnell, 1964) and examined for skeletal defects. The remaining two-thirds of the foetuses were examined for soft-tissue anomalies, either by histological methods or by freehand sectioning (Wilson, 1965).

In addition to the post weaning F1b rats, selected organs from 5 dams from each treatment group were evaluated histologically during each teratological phase.

Dose / conc.:

0.1 other: %

Remarks:

nominal in diet; equivalent to 112 mg/kg bw/day

Dose / conc.:

0.5 other: %

Remarks:

nominal in diet; equivalent to 447 mg/kg bw/day

No. of animals per sex per dose:

22 (F0)
25 (F1b) - 20 for breeding F2 generation and 5 for histological examination

Control animals:

yes, plain diet

Parental animals: Observations and examinations:

Feed consumption and body weights were recorded at weekly intervals.
Evaluation of conception rate, number of stillborn, number of born per litter, number of weaned per litter

Oestrous cyclicity (parental animals):

Pregnancies were timed by vaginal smearing (Long and Evans, 1922)

Litter observations:

All newborns were counted at birth, but not handled until they were 4 days old. They were then weighed, sexed and inspected grossly.
- F1a: Discarded
- F1b: 5 per sex were necropsied and examined histologically.
- F1c: Teratologic evaluation (one-half of each treatment group on day 13, the other half on day 21 of gestation period). The pregnant females were examined for number of resorptions, corpora lutea and implantations
- F2a: Discarded
- F2b: Teratologic evaluation (one-half of each treatment group on day 13, the other half on day 21 of gestation period). The pregnant females were examined for number of resorptions, corpora lutea and implantations.

Postmortem examinations (parental animals):

Pregnant females were examined for number of resorptions, corpora lutea and implantations

Postmortem examinations (offspring):

Teratologic evaluation: Foetuses were examined for skeletal defects, soft-tissue defects, gross abnormalities

Statistics:

Analyses of variance were done on the appropriate data (Snedecor, 1946), and the partitioning was done by the Tukey "minimum difference" test as described by Scheffe (1952).

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality was observed in four females, two were in the group administered 0.5% HEDP-2Na continuously and two were from the group receiving the high level during organogenesis. The cause of death could not be determined in two cases. However, death of the other two was attributed to pneumonia in one and to thyroid tumour in the other.
A female administered 0.5% HEDP-2Na during gestation, died on day 25 of the third phase, 2 days after giving birth to 7 dead pups. Five more pups were found in the uterus at necropsy. One of these was exencephalic and four were hydroencephalic; they could not be inspected further because of decomposition.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The weight gains were similar for all dose groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No differences were observed between treated and control animals.

Food efficiency:

no effects observed

Description (incidence and severity):

No differences were observed between treated and control animals.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No differences were observed in the growth between treated and control animals.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The overall conception rate of 90% shows that the compound did not interfere with implantation. Two litters, with an indeterminate number of pups, were stillborn in the first litters; one each in females administered 0.5 or 0.1% HEDP-2Na continuously. The former had a normal second litter, but the latter did not become pregnant again.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The overall conception rate of 90% shows that the compound did not interfere with the spermatogenesis.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

An increase in the number of stillborn foetuses of P1 litters were observed in dams administered 0.5% HEDP-2Na. Dams receiving 0.1% HEDP-2Na only during pregnancy had significantly more pups than either control rats or those administered the same level continuously. There were no significant differences in the numbers of resorptions or of implantations in females sacrificed at 13 days postcoitum; corpora lutea were not counted in these females.

Dose descriptor:

NOAEL

Effect level:

447 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed

Remarks on result:

other: equivalent to 368 mg/kg bw/day active acid

Critical effects observed:

not specified

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

There was a significant reduction in the number of live pups born to dams administered 0.5% HEDP-2Na during organogenesis in the F1a phase and an increase in the number of stillborn foetuses, although these dams had a higher average number of pups than the control group.

In the P1 phase, those dams receiving 0.1% HEDP-2Na only during pregnancy had significantly more pups than either control rats or those administered the same level continuously. There were no significant differences in the preweaning mortality. Two litters, with an indeterminate number of pups, were stillborn in the first litters; one each in females administered 0.1 or 0.5 % HEDP-2Na continuously. The former had a normal second litter, but the latter did not become pregnant again. A female administered 0.5% HEDP-2Na during gestation, died on day 25 of the third phase, 2 days after giving birth to 7 dead pups. Five more pups were found in the uterus at necropsy. One of these was exencephalic and four were hydroencephalic; they could not be inspected further because of decomposition.

There was no significant mortality in rat embryos from the females sacrificed at 13 days in either generation.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The weight gains were similar for all dose groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No differences were observed between treated and control animals.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not specified

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Three malformed foetuses were found in 42 litters (0.6% incidence); 1 of these was from a female administered 0.1% HEDP-2Na continuously (left 12th rib missing) and 2 were from a female administered 0.5% of the test material during organogenesis (hydroencephalocele in 1, sternoschisis in the other). However, these changes were not considered as treatment-related.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No differences were observed in the growth between treated and control animals. There was a significant reduction in the number of live pups born to dams fed 0.5% of HEDP-2Na during organogenesis in the F1a phase although these dams had a higher average number of pups than the control mothers. The overall conception rate of 90% shows that the compound did not interfere with the spermatogenesis or nidation. The number of pups born and weaned were comparable to historical controls.

Teratologic evaluation of the third litters (F1c) showed no significant treatment differences in the number of live foetuses, corpora lutea, or implantations in females sacrificed at 21 days postcoitum. However, significantly more resorptions occurred in the control (untreated) group. In addition, 7 rats scattered among the test and control group resorbed all their embryos.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

447 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed

Remarks on result:

other: equivalent to 368 mg/kg bw/day active acid).

Critical effects observed:

not specified

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

No significant mortality in rat embryos was observed from the females sacrificed at 13 days in either generation. However, in the second generation, there were fewer foetuses in the females administered 0.5% HEDP-2Na. Although varying statistical evidence, these collective data indicate that the 0.5% dose level of is somewhat toxic to the embryo and that it is slightly more toxic when provided only during pregnancy, rather than when administered chronically.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Second-generation foetuses were slightly heavier than those born in the first generation, however, the weights were not significantly different among treatment groups.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

The first litters of the second generation females were smaller than the previous generation, however, there were no significant differences in any of the parameters among the groups. Overall, 1.2% of the 1028 foetuses examined were defective, including 2.5% of the 211 foetuses from the control group. No defect was preponderant. Some variation was observed in the incidence of extra ribs and the number of sternebrae, however, the incidences of both were low and scattered randomly throughout both control and experimental groups.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

In the second litters used for teratologic evaluation, there were no significant differences observed among corpora lutea, implantations or resorptions at any time periods. At 21 days, the number of implantations was reduced in rats administered 0.5% HEDP-2Na continuously but was statistically different only from those administered 0.1% of the test substance during organogenesis. Corpora lutea formation in rats administered 0.5% HEDP-2Na continuously and sacrificed at 21 days was significantly depressed as compared with control animals. There was a decrease in the number of live foetuses born to both groups of dams receiving 0.5% of the test substance, however, the decrease was significant only in the group treated only during gestation.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

447 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Remarks on result:

other: equivalent to 368 mg/kg bw/day active acid.

Critical effects observed:

not specified

Reproductive effects observed:

not specified

TABLE 1: EFFECTS ON THE REPRODUCTIVE PERFORMANCE OF PARENT GENERATION (F0) RATS AND THEIR FIRST TWO LITTERS (F1a and F1b)

 

Group	No. pregnant		Conception rate		No. of stillborn		Av. No. born per litter		Av. No. weaned per litter		Av. weaning weight (g)
	F1a	F1b	F1a	F1b	F1a	F1b	F1a	F1b	F1a	F1b	F1a	F1b
Untreated control	17	19	85	95	2	6	13.0	10.4	7.6***	7.1***	47.6	46.9
0.5% continuously	18	17	90	89.5	2	9	12.6	12.8	7.9	7.5	47.3	44.3
0.1% continuously	18	16	90	80	4	10	12.2	10.2	7.8	7.0	44.8	47.4
0.5% day 6-15 postcoitum	19	18	95	94.7	5	22	9.8*	12.7	7.1	7.6	47.1	48.3
0.1% day 6-15 postcoitum	19	18	95	90	2	15	12.7	13.5**	7.5	7.9	46.9	44.8

 

* Significantly different from control rats (p<0.05)

** Significantly different from control rats and those fed 0.1% continuously (p<0.05)

*** All litters containing more than 8 pups were reduced to that number at 4 d

 

 

TABLE 2: EFFECTS ON THE REPRODUCTION OF PARENT GENERATION (F0) RATS AND ON THEIR THIRD LITTERS (F1C)

 

Effect	Control		0.5% continuously		0.1% continuously		0.5% days 6-15 postcoitum		0.1% days 6-15 postcoitum
	13 d	21 d	13 d	21 d	13 d	21 d	13 d	21 d	13 d	21 d
No. pregant	9	10	10	8*	9	9	9*	8*	9	7
% pregnant	90.0	100.0	100.0	88.9	90.0	90.0	100.0	88.9	90.0	70.0
No. total resorptions	1	2	1	0	1	0	1	0	1	0
Av. No. corpora lutea	-	15.3	-	14.0	-	14.3	-	12.8	-	15.5
Av. No. implantations	13.0	13.4	14.3	14.4	12.8	13.6	16.3	13.4	13.3	14.4
Av. No. resorptions	2.0	3.9**	0.3	0.0	1.3	0.0	1.8	0.0	0.6	0.0
Av. No. live fetuses	-	11.6	-	14.4	-	14.3	-	13.4	-	14.4
No. dead fetuses	-	0	-	0	-	0	-	1	-	0
No. fetuses abnormalities	-	0	-	0	-	1	-	2	-	0
Av. weight fetuses (g)	-	3.5	-	3.5	-	3.9	-	3.5	-	3.7

 

* Deaths earlier had reduced these groups in number

** Significantly different from other females sacrificed at 21 d (p<0.05)

 

 

TABLE 3: EFFECTS ON THE REPRODUCTION OF SECOND GENERATION PARENTS (F1b) AND ON THEIR FIRST LITTERS (F2a)

 

	Control	0.5% continuously	0.1% continuously	0.5% days 6-15 postcoitum	0.1% days 6-15 postcoitum
No. pregant	19	15	20	19	18
% pregnant	95.0	75.0	100.0	95.0	90.0
Av. No. born live per litter	10.2	9.1	8.9	9.1	10.3
Av. pup weight at 4d (g)	9.7	9.5	10.4	10.1	9.6
Av. No. weaned per litter*	7.1	7.4	6.7	7.0	7.4
Av. weight of pups  at weaning (g)	43.0	41.7	46.0	45.5	42.9

 

* Litters containing more than 8 pups were reduced to that number at 4 d

 

 

 

TABLE 4: EFFECTS ON THE REPRODUCTION OF SECOND GENERATION PARENTS (F1b) AND ON THEIR SECOND LITTERS (F2b)

 

Effect	Control		0.5% continuously		0.1% continuously		0.5% days 6-15 postcoitum		0.1% days 6-15 postcoitum
	13 d	21 d	13 d	21 d	13 d	21 d	13 d	21 d	13 d	21 d
No. pregant	10	10	10	7	10	9	9	9	10	10
% pregnant	100.0	100.0	100.0	70.0	100.0	90.0	90.0	90.0	100.0	100.0
No. total resorptions	0	1	1	0	1	0	2	1	1	0
Av. No. corpora lutea	13.9	14.2	13.6	11.9*	13.6	13.6	13.4	13.4	13.2	13.8
Av. No. implantations	14.3	13.1	12.0	10.7**	13.0	13.4	13.3	11.2	13.0	13.5
Av. No. resorptions	1.2	1.4	0.9	0.9	1.8	0.9	1.9	3.8	1.3	0.9
Av. No. live fetuses	-	13.0	-	9.9	-	12.4	-	9.1***	-	12.6
No. dead fetuses	-	0	-	0	-	0	-	0	-	0
No. fetuses abnormalities	-	5	-	1	-	1	-	0	-	3
Av. weight fetuses (g)	-	4.8	-	5.4	-	5.4	-	4.9	-	5.2

 

*Significantly less than control rats at 21 d (p<0.05)

** Significantly less than rats fed 0.1% days 6-15 postcoitum (p<0.05)

*** Significantly less than control rats (p<0.05)

 

 

TABLE 5: INCIDENCES AND DISTRIBUTION OF FETAL ABNORMALITIES OBSERVED IN RATS

 

	Control		0.5% continuously		0.1% continuously		0.5% days 6-15 postcoitum		0.1% days 6-15 postcoitum
	F1c	F2b	F1c	F2b	F1c	F2b	F1c	F2b	F1c	F2b
No. litters	7	9	8	7	9	9	7	8	7	10
No. fetuses examined	93	118	115	69	122	113	94	74	102	128
No. fetuses examined for skeletal defects	31	37	38	22	39	38	30	24	33	43
No. fetuses examined for soft-tissue defects	62	81	77	47	83	75	64	50	69	85
Fetuses with gross abnormalities (%)	0	2.5*	0	0	0	0	1.1	0	0	0
Fetuses with skeletal defects (%)	0	0	0	0	2.5~	0	3.3	0	0	0
Fetuses with soft-tissue defects (%)	0	5.0#	0	2.1	0	1.3	1.1	0	0	3.5
Facial dysgenesis	-	1.7	-	-	-	-	-	-	-	-
Hematoma	-	0.8	-	-	-	-	-	-	-	-
Hydronephrosis	-	2.5	-	-	-	1.3	-	-	-	1.2
Double aorta	-	1.2	-	-	-	-	-	-	-	-
Cryptorchism	-	-	-	2.1	-	-	-	-	-	-
Hydro-encephalocele	-	-	-	-	-	-	1.1	-	-	-
Ascites	-	-	-	-	-	-	-	-	-	1.2
Hyperplastic testicle	-	1.2	-	-	-	-	-	-	-	-
Abdominal hemorrhaging	-	-	-	-	-	-	-	-	-	1.2
Missing rib, L 12th	-	-	-	-	2.5	-	-	-	-	-
Sternoschisis	-	-	-	-	-	-	1.1	-	-	-

 

* Percentages are based upon total number of fetuses examined

~ Percentages are based upon number cleared and stained

# Percentages are based upon number of animals examined for soft-tissue effects

Conclusions:

In a two-generation feeding study (reliability score 2), conducted to a protocol similar to OECD Test Guideline 416 and pre-GLP, HEDP (2-3Na) was administered to rats in doses of 0, 0.1 or 0.5 % (equivalent to 0 mg/kg bw/day, approximately 112 mg/kg bw/day and approximately 447 mg/kg bw/day respectively), either continuously to both sexes and to females only on Gestation Day 6-15. An impairment of reproductive function was not observed in male or female rats at relatively high dose levels. No treatment-related malformations or developmental variations were noted at any exposure level. Mean numbers of corpora lutea, implantation sites, resorptions, viable foetuses and mean foetal weights were unaffected by treatment at an exposure level of 112 mg/kg bw/day. In the second generation, mortality was observed at the highest dose tested. It was concluded that a NOAEL for maternal dams, the F1 and F2 generations are 447 mg/kg bw/day (equivalent to 368 mg/kg bw/day active acid).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

368 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Reliability score 2

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In the key two-generation feeding study (reliability score 2), conducted to a protocol similar to OECD Test Guideline 416 and pre-GLP, HEDP (2-3Na) was administered to rats in doses of 0, 0.1 or 0.5% (equivalent to 0 mg/kg bw/day, approximately 112 mg/kg bw/day and approximately 447 mg/kg bw/day respectively), either continuously to both sexes and to females only on Gestation Day 6-15. An impairment of reproductive function was not observed in male or female rats at relatively high dose levels. No treatment-related malformations or developmental variations were noted at any exposure level. Mean numbers of corpora lutea, implantation sites, resorptions, viable foetuses and mean foetal weights were unaffected by treatment at an exposure level of 112 mg/kg bw/day. It was concluded that a NOAEL for maternal dams and for the F1and F2 generations are 447 mg/kg bw/day which is equivalent to 368 mg/kg bw/day active acid (Procter & Gamble, 1971a).

See read-across justification in IUCLID Section 13.

Effects on developmental toxicity

Description of key information

There are no developmental toxicity data for HEDP-xK, therefore good quality data are read-across from the category member HEDP (2 -3Na).

In a two-generation feeding study (reliability score 2), conducted to a protocol similar to OECD Test Guideline 416 and pre-GLP, HEDP (2-3Na) was administered to rats in doses of 0, 0.1 or 0.5 % (equivalent to 0 mg/kg bw/day, approximately 112 mg/kg bw/day and approximately 447 mg/kg bw/day respectively), either continuously to both sexes and to females only on Gestation Day 6-15. No treatment-related malformations or developmental variations were noted at any exposure level. A NOAEL for maternal animals was concluded to be 112 mg/kg bw/day (equivalent to 93 mg/kg bw/day active acid) and a NOAEL for foetuses was concluded to be 447 mg/kg bw/day (equivalent to 368 mg/kg bw/day active acid) (Procter & Gamble, 1971a).

 

In the key developmental toxicity study (reliability score 2), conducted according to OECD Test Guideline 414 and pre-GLP, HEDP (2 -3Na) was administered to in concentrations of 0, 25, 50 or 100 mg/kg bw/day to 20 New Zealand rabbits. A maternal and developmental NOAEL of ≥ 100 mg/kg bw/day is concluded for rabbits (Procter & Gamble, 1971a).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

Dosing from day 2-16 instead of day 6-18; uterine weights (does) and sex ratios (offspring) were not evaluated

GLP compliance:

no

Remarks:

prior to GLP

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
Virgin New Zealand rabbits, 5-6 months of age and about 4 kg bodyweight at the time of insemination, were randomly distributed into groups of 25 (pre-study) or 20 (main study) does on the basis of weight and litter after an 18-day holding period. Water and food (Purina Rabbit Chow) was available ad libitum. They were housed in standard stainless steel rabbit cages, one female to a cage.

ENVIRONMENTAL CONDITIONS:
Room temperature was maintained at 23 +/- 1°C, and relative humidity at 50 +/- 5%. Lighting was on a 12 hour cycle and background music was employed to equalize ambient noises.

Route of administration:

other: oral/gavage in the pre-study, oral/feed and oral/gavage in the main study

Vehicle:

other: drinking water (gavage application, pre-study and main study), no vehicle (incorporation in food, main study)

Details on exposure:

PRE-STUDY:
Except for the untreated control group, the females were dosed with either the test material in water or water alone on days 2 through 16 (day inseminated = day 1). Each doe recieved 2 mL of fluid per kg bw. Dosing was commenced prior to implantation (day 7) so that any possible preimplantation effects might be revealed. The compound was given as an aqueous mixture by intubation.

MAIN STUDY:
In the main study, doses of 25, 50 or 100 mg/kg bw/day were used. The compound was incorporated into ground rabbit feed which was then repelleted and fed to the rabbits from day 2 through day 16 of pregnancy.
To determine whether the ingestion of HEDP-2Na in the feed might cause different effects than when introduced by gavage, another group of rabbits was given 100 mg/kg bw/day of the material by stomach tube just as was done in the Pre-study. Control groups, untreated and water-treated, were included.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Females were artificially inseminated by the method of Gibson et al (1966).

Duration of treatment / exposure:

Gestation Day 2-16

Frequency of treatment:

Daily

Duration of test:

29 days

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control group

Dose / conc.:

25 mg/kg bw/day

Dose / conc.:

50 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

No. of animals per sex per dose:

Pre-study: 25
Main study: 20

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

PRE-STUDY:
The females were artificially inseminated by the method of Gibson et al (1966) and except for the untreated controls, were dosed with either the test material in water or water alone on days 2 through 16 (day inseminated = day 1). Each doe recieved 2 mL of fluid per kg bw. Dosing was commenced prior to implantation (day 7) so that any possible preimplantation effects might be revealed. The compound was given as an aqueous mixture by intubation. Mortality was observed in animals receiving 500 mg/kg bw/day after day 4 or 5 of dosing. Consequently, the 500 mg/kg bw/day dosage was reduced to 250 mg/kg bw/day. Pregnant animals were sacrificed on day 29 and examined for resorptions, corpora lutea and implantations. The foetuses were dried of amniotic fluid, sexed, carefully inspected for gross abnormalities and weighed. One-third of the fetuses were cleared, stained with Alizarin red stain (Staples and Schnell, 1964) and examined for skeletal defects. The remaining two-thirds of the fetuses were examined for soft-tissue anomalies, either by histological methods or by freehand sectioning (Wilson, 1965).

MAIN STUDY:
Due to observed maternal toxicity from the 500 mg/kg bw/day dose of HEDP-2Na in the pre-study, doses of 25, 50 and 100 mg/kg bw/day were used. The compound was incorporated into ground rabbit feed (Purina Rabbit Chow) which was then repelleted and fed to the rabbits from day 2 through day 16 of pregnancy.
To determine whether the ingestion of HEDP-2Na in the feed might cause different effects than when introduced by gavage, another group of rabbits was given 100 mg/kg bw/day of HEDP-2Na by a stomach tube, similarly performed as in the pre-study. Control groups, untreated and water-treated animals were included.
To reduce the possibilty of a dietary bias, all rabbits were fed ground feed which had been repelleted during both the orientation period and the 29 days of gestation.The feed consumed by 25 animals was measured for 14 days during the orientation period to establish the dietary level of test substance to be incorporated into the diet. Additionally, the feed consumed from day 2 through day 16 of pregnancy was measured for all animals to determine the actual amount of HEDP-2Na ingested as well as determine whether the stress of intubing the rabbits affected feed intake.
All animals were inseminated by the method of Gibson et al., 1966 and weighed on that day and again on day 29 of pregnancy. The former weights were used to calculate the dose levels, whether given by intubation or incorporated into the feed. Those given the HEDP-2Na by stomach tube and water-treated controls were weighed daily, since this caused no additional trauma, in order to monitor the weight gains throughout gestation.
Likewise, the laparotomies and collection of data were done as described in the pre-study.

Maternal examinations:

Pregnant does were examined for resorptions, corpora lutea and implantations. The body weight was recorded on GD0 and GD29. The food intake was recorded daily.

Fetal examinations:

The foetuses were dried of amniotic fluid, sexed, carefully inspected for gross abnormalities and weighed. One-third of the foetuses were cleared, stained with Alizarin red stain (Staples and Schnell, 1964) and examined for skeletal defects. The remaining two-thirds of the foetuses were examined for soft-tissue anomalies, either by histological methods or by freehand sectioning (Wilson, 1965).

Statistics:

Analyses of variance were done on the appropriate data (Snedecor, 1946), and the partitioning was done by the Tukey "minimum difference" test as described by Scheffe (1952).

Clinical signs:

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality was observed in the pre-study in animals receiving 500 mg/kg bw/day after day 4 or 5 of dosing. Four females that received only 3 doses of 500 mg/kg bw/day before having the dose reduced to 250 mg/kg bw/day survived to term. They were not included in the statistical analyses, however, their data were included so limited comparisons between the two doses could be performed.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the gain in bodyweight among the rabbits. The group intubated with 100 mg/kg bw/day of HEDP-2Na consumed the least amount and gained the least amount of weight during gestation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the feed consumption among the rabbits.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the pre-study, renal tubular degeneration was observed in all dams administered 250 and 500 mg/kg bw/day as well as in 20% of those dosed with 100 mg/kg bw/day. However, control animals were similarly affected (occurrence of 50%) and therefore, it was not considered as treatment-related effects.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

One dam administered 100 mg/kg bw/day of HEDP-2Na by a stomach tube, aborted at 23 days and the foetuses were dead. However, this was attributed to severe respiratory disease and is considered not as a treatment-related effect.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no significant differences in implantations.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no significant differences in resorptions.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no significant differences in resorptions.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

In the pre-study, New Zealand rabbits were successfully inseminated in 81% of the attempts, although this varied significantly from 100% in the water-dosed control to 68% in the group treated with 100 mg/kg bw/day of HEDP-2Na. In the main study, the overall conception rate for the 120 rabbits was 92.5% and varied from 85% in the non-treated control group to 100% in the water-treated controls. The conception rates for the HEDP-2Na-treated groups were 90% or 95% respectively, indicating the test material had no effect on conception or on nidation.

Other effects:

no effects observed

Description (incidence and severity):

No significant differences in the numbers of corpora lutea was observed.

Dose descriptor:

NOAEL

Effect level:

>= 100 mg/kg bw/day

Basis for effect level:

other: No adverse effects observed

Remarks on result:

other: equivalent to 82.4 mg/kg bw/day active acid

Abnormalities:

not specified

Fetal body weight changes:

not examined

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

In the pre-study, the number of live foetuses was reduced in the group administered 100 mg/kg bw/day, however, this was not significant and was not observed in the main study.

Changes in sex ratio:

not examined

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

In the pre-study, no significant differences in litter weights were observed. In the main study, foetuses from dams administered 100 mg/kg bw/day of HEDP-2Na daily by gavage, were significantly smaller than those from untreated control dams. However, they were from slightly larger litters. Their weights were not significantly different from the water-dosed control foetus weights. The difference might have been caused by the stress of intubation of the dam. Additionally, the reduced weight was within the normal variation and consequently, not considered as treatment related.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

In the pre-study, no defective foetuses (18 in total) were observed in dams intubated with higher doses of the test substance. Totally, 2 of 99 foetuses from dams intubated with 100 mg/kg bw/day were defective, however, this was considered as incidental and not as treatment-related effects. Of the 868 rabbit foetuses examined in the main study, 17 (less than 2%), were defective and the treated groups were not significantly different from the controls. Spina bifida and folded retina were the most frequent occurring defects.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Totally, 30 to 40% of all foetuses in the pre-study had supernumerary ribs; most of these were bilateral. Variations in the sternebrae occurred; atrophy of the fifth sternebrae was most frequently observed. Intubation or administration of the test substance alone or in combination has affected the incidence of rib or sternal variations.

Visceral malformations:

not examined

Other effects:

not examined

Dose descriptor:

NOAEL

Effect level:

>= 100 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: equivalent to 82.4 mg/kg bw/day active acid

Abnormalities:

not specified

Developmental effects observed:

not specified

TABLE 1 (PRE-STUDY): EFFECTS OF INTUBATED HEDP-2Na ON REPRODUCTION AND TERATOGENY OF NEW ZEALAND RABBITS

 

	Untreated control	H2O control	100 mg/kg	500 mg/kg****
No. pregnant	19	22	15	-
Conception rate	82.6	100.0	68.2***	-
No. females dead	1	1	2	20
Mean corpora lutea	9.7	9.6	10.3	11.3
Mean resorptions	1.2	1.1	1.4	3.3
Mean live fetuses	8.0	7.8	6.7	9.0
Mean fetus weights (g)	26.4	25.8	26.3	-
No. fetuses examined	146	158	99	18
No. with soft-tissue defects	1	6	2	0
No. with skeletal defects	0	0	0	0
Defective fetuses (%) *	0.6	3.9	2.0	0
Hydronephrosis	-	2.0**	-	-
Herniated lens and folded retina	1.1	-	-	-
Aortic arch stenosis	-	2.0	-	-
Missing right kidney and ureter	-	1.0	-	-
Cor biloculare	-	1.0	-	-
Testicular atrophy	-	1.0	-	-
Hydroencephaly	-	-	1.5	-
Spina bifida	-	-	1.5	-

 

* Based on total number of fetuses examined

** More than one defect may have appeared in 1 fetus

*** Significantly less than H2O dosed control (p<0.05)

**** The dosage was reduced to 250 mg/kg, but most died. Four survived: two were sacrificed early and two completed pregnancies. Their values were not included in statistical analyses.

 

TABLE 2 (MAIN STUDY): EFFECTS OF HEDP-2Na ON THE REPRODUCTION AND TERATOGENY OF NEW ZEALAND RABBITS

 

	Untreated control	H2O control	25 mg/kg	50 mg/kg	100 mg/kg	100 mg/kg by gavage
No. pregnant	17	20	18	19	19	18
Conception rate	85	100	90	95	95	90
Mean corpora lutea	10.8	10.1	9.9	9.7	10.4	11.6
Mean resorptions	0.9	0.9	0.9	1.8	1.2	0.9
Mean live fetuses	7.8	8.0	8.5	7.1	8.4	9.4
Mean fetus weights (g)	32.0	30.8	30.9	30.0	28.0	27.3*
No. fetuses examined	127	155	151	134	156	145
No. with soft-tissue defects	2	4	1	1	4	4
No. with skeletal defects	0	1	0	0	0	0
Defective fetuses (%) **	1.6	3.2	0.7	0.8	2.6	2.8
Spina bifida	-	1.9	0.7	-	0.6	-
Hydroencephaly	-	0.6	-	-	0.6	-
Testicular atrophy	0.8	-	-	-	-	0.7
Cryptorchism	0.8	-	-	-	-	-
Folded retina	0.6	-	-	0.8	0.6	-
Coloboma	-	0.6	-	-	-	-
Anopthalmia	-	-	-	-	0.6***	-
Arrhinencephalia	-	-	-	-	0.6	-
Bilateral hydronephrosis	-	-	-	-	0.6	-
Gastroschisis	-	-	-	-	0.6	-
Coarctation of aorta	-	-	-	-	-	0.7

 

* Significantly less than the nontreated control group only (p<0.05)

** Based upon total number examined

*** More than one defect may have been present in a fetus

 

Conclusions:

In a developmental toxicity study (reliability score 2), conducted according to OECD Test Guideline 414 and pre-GLP, HEDP (2-3Na) was administered to in concentrations of 0, 25, 50 or 100 mg/kg bw/day to 20 New Zealand rabbits. A maternal and developmental NOAEL of ≥ 100 mg/kg bw/day is concluded for rabbits.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

Use of 2 dose levels; Sexual milestones in pups/analytical confirmation of dose not given; No sperm analyses (but conception rate of 90% suggests unaffected spermatogenesis, and OECD 416 allows omission if information is available from e.g. 90 day study)

GLP compliance:

no

Remarks:

prior to GLP

Limit test:

no

Species:

rat

Strain:

other: Charles-River

Details on test animals or test system and environmental conditions:

Weanling Charles-River rats were distributed into 5 groups, each composed of 22 females and 22 males, according to body weight and litter. They were housed in stainless steel cages with food (Ground Purina Laboratory Chow) and fresh water furnished ad libitum. Room temperature was maintained at 23 +/- 1°C, and relative humidity at 50 +/- 5%. Lighting was on a 12 hour cycle and background music was employed to equalize ambient noises. During the first 8 weeks, the rats were caged individually and feed consumption and body weights were recorded at weekly intervals. The rats were then paired and placed into mating cages. This procedure was repeated for the breeders in the second generation (F1b). Pregnant females were placed in nesting cages which were furnished with a plywood liner and shredded paper.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

When appropriate, HEDP-2Na was mixed with the diet at levels of 0.5 and 0.1%. Both sexes were fed with the respective diets either continuously or only during the time when the females were in their sixth through fifteenth days of gestation.
HEDP-2Na was either administered continuously from weaning (week 5) to both sexes, or to pregnant females on GD 6-15 at dose levels of 0, 0.1% or 0.5% in diet (Ground Purina Laboratory Chow). Doses stated to represent 1/12 and 1/3 of the LD50 dose (1.34 g/kg) hence target doses presumed to be 112 mg/kg bw and 447 mg/kg bw.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

One male, one female placed in mating cages. Start of pregnancy determined from positive vaginal smear.

Duration of treatment / exposure:

Exposure period: continuous or Gestation Day 6-15

Frequency of treatment:

daily

Duration of test:

two-generation study

Dose / conc.:

0.1 other: %

Remarks:

nominal in diet

Dose / conc.:

0.5 other: %

Remarks:

nominal in diet

No. of animals per sex per dose:

22 (F0)
25 (F1b) - 20 for breeding F2 generation and 5 for histological examination

Control animals:

yes, plain diet

Details on study design:

When appropriate, HEDP-2Na was mixed with the diet at levels of 0.5 and 0.1%. Both sexes were fed with the respective diets either continuously or only during the time when the females were in their sixth through fifteenth days of gestation. During the first 8 weeks, the rats were caged individually and feed consumption and body weights were recorded at weekly intervals. The rats were then paired and placed into mating cages. This procedure was repeated for the breeders in the second generation (F1b). Pregnancies were timed by vaginal smearing (Long and Evans, 1922), and identification of sperm in the smear designated day 0. The pregnant females were then placed in nesting cages which were furnished with a plywood liner and shredded paper.

The original females (F0) were allowed to deliver their first 2 litters, while the third litters were used for teratologic evaluation. The newborn were counted at birth, but not handled until they were 4 days old. They were then weighed, sexed and inspected grossly. All litters containing more than 8 pups were reduced to that number to equalize the stress on the mothers during the lactation period. After the weaning weights were recorded, all F1a pups were discarded, but 25 weanlings of each sex, from each treatment group, were selected from the F1b litters for second generation breeding stock.

20 pairs of rats from each group were bred and the remaining 5 pairs were necropsied and examined histologically. Subsequently, the first litters of the second generation (F2a) were evaluated similarly to the F1a pups, while the second litters (F2b) were used for teratologic evaluation. During the teratological phases one-half of each treatment group, selected prior to mating, were sacrificed by excessive ether inhalation on day 13 and the other half on day 21 of the gestation period.

The pregnant females were examined for number of resorptions, corpora lutea and implantations. The fetuses were dried of amniotic fluid, sexed, carefully inspected for gross abnormalities and weighed. One-third of the fetuses were cleared, stained with Alizarin red stain (Staples and Schnell, 1964) and examined for skeletal defects. The remaining two-thirds of the fetuses were examined for soft-tissue anomalies, either by histological methods or by freehand sectioning (Wilson, 1965).

In addition to the post weaning F1b rats, selected organs from 5 dams from each treatment group were evaluated histologically during each teratological phase.

Maternal examinations:

Teratologic evaluation: Pregnant females were examined for number of resorptions, corpora lutea and implantations.

Fetal examinations:

Teratologic evaluation: Fetuses were examined for skeletal defects, soft-tissue defects, gross abnormalities

Statistics:

Analyses of variance were done on the appropriate data (Snedecor, 1946), and the partitioning was done by the Tukey "minimum difference" test as described by Scheffe (1952).

Historical control data:

In the naturally born litters, the number of pups born and weaned were comparable to those seen in previous breeding studies from this laboratory (Nolen and Alexander, 1966).

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality was observed in four females, two were in the group administered 0.5% HEDP-2Na continuously and two were from the group receiving the high level during organogenesis. The cause of death could not be determined in two cases. However, death of the other two was attributed to pneumonia in one and to thyroid tumour in the other.
A female administered 0.5% HEDP-2Na during gestation, died on day 25 of the third phase, 2 days after giving birth to 7 dead pups. Five more pups were found in the uterus at necropsy. One of these was exencephalic and four were hydroencephalic; they could not be inspected further because of decomposition.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The weight gains were similar for all dose groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No differences were observed between treated and control animals.

Food efficiency:

no effects observed

Description (incidence and severity):

No differences were observed between treated and control animals.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

not examined

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

There were no significant differences in the numbers of implantations in females sacrificed at 13 days postcoitum. Teratologic evaluation of the third litters (F1c) showed no significant treatment differences in the implantations in females sacrificed at 21 days postcoitum. At 21 days, the number of implantations was reduced in rats administered 0.5% HEDP-2Na continuously, however, was only statistically different from those administered 0.1% during organogenesis.

Total litter losses by resorption:

not specified

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no significant differences in the numbers of resorptions, however, significantly more resorptions occurred in the control group. In total, 7 rats scattered among the test and control group resorbed all their embryos.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

There was a significant reduction in the number of live pups born to dams administered 0.5% of HEDP-2Na during organogenesis in the F1a phase and an increase in the number of stillborn foetuses of F1b litters, although these dams had a higher average number of pups than the control dams. In the F1b phase, dams receiving 0.1% of HEDP-2Na only during pregnancy had significantly more pups than either control rats or those administered the same level continuously. There were no significant differences in the preweaning mortality.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Corpora lutea were not counted in females sacrified at 13 days postcoitum. Teratologic evaluation of the third litters (F1c) showed no significant treatment differences in the corpora lutea. Corpora lutea formation in rats administered 0.5% of HEDP-2Na continuously (sacrificed at 21 days) was significantly depressed as compared with control animals.

Dose descriptor:

NOAEL

Effect level:

ca. 447 mg/kg bw/day

Basis for effect level:

other: No adverse effects observed

Abnormalities:

not specified

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The weight gains were similar for all dose groups.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

Two litters, with an indeterminate number of pups, were stillborn in the first litters; one each in females administered 0.5 or 0.1% of HEDP-2Na continuously. The former had a normal second litter, but the latter did not become pregnant again. Teratologic evaluation of the third litters (F1c) showed no significant treatment differences in the number of live foetuses. There was a decrease in the number of live foetuses born to both groups of dams receiving 0.5% of the test substance, however, the decrease was significant only in the group treated during gestation.

Changes in sex ratio:

not examined

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Second-generation foetuses were slightly heavier than those born in the first generation, although the first litters of the second generation females were smaller than the previous generation. However, the weights were not significantly different among treatment groups.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

In the naturally born litters, the number of pups born and weaned were comparable to historical control data.

External malformations:

no effects observed

Description (incidence and severity):

One foetus had hydroencephalocele and one foetus had sternoschisis and both were delivered from a dam administered 0.5% of HEDP-2Na during organogenesis. A female administered 0.5% of HEDP-2Na during gestation, died on day 25 of the third phase and five pups were found in the uterus at necropsy. One of these was exencephalic and four were hydroencephalic. The foetuses could not be inspected further because of decomposition.
The incidence of malformed foetuses were 0.6 % without any effects being preponderant and therefore, considered as incidental and is not considered as treatment-related effects.

Skeletal malformations:

no effects observed

Description (incidence and severity):

One foetus had the left 12th rib missing, delivered from a mother administered 0.1% of HEDP-2Na continuously. However, the incidence of malformed foetuses were 0.6 % and therefore, considered as incidental and is not considered as treatment-related effects.

Visceral malformations:

not examined

Other effects:

not examined

Dose descriptor:

NOAEL

Effect level:

ca. 447 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Remarks on result:

other: equivalent to 368 mg/kg bw active acid

Abnormalities:

not specified

Developmental effects observed:

not specified

TABLE 1: EFFECTS ON THE REPRODUCTIVE PERFORMANCE OF PARENT GENERATION (F0) RATS AND THEIR FIRST TWO LITTERS (F1a and F1b)

 

 

Group	No. pregnant		Conception rate		No. of stillborn		Av. No. born per litter		Av. No. weaned per litter		Av. weaning weight (g)
	F1a	F1b	F1a	F1b	F1a	F1b	F1a	F1b	F1a	F1b	F1a	F1b
Untreated control	17	19	85	95	2	6	13.0	10.4	7.6***	7.1***	47.6	46.9
0.5% continuously	18	17	90	89.5	2	9	12.6	12.8	7.9	7.5	47.3	44.3
0.1% continuously	18	16	90	80	4	10	12.2	10.2	7.8	7.0	44.8	47.4
0.5% day 6-15 postcoitum	19	18	95	94.7	5	22	9.8*	12.7	7.1	7.6	47.1	48.3
0.1% day 6-15 postcoitum	19	18	95	90	2	15	12.7	13.5**	7.5	7.9	46.9	44.8

 

* Significantly different from control rats (p<0.05)

** Significantly different from control rats and those fed 0.1% continuously (p<0.05)

*** All litters containing more than 8 pups were reduced to that number at 4 d

 

 

TABLE 2: EFFECTS ON THE REPRODUCTION OF PARENT GENERATION (F0) RATS AND ON THEIR THIRD LITTERS (F1C)

 

Effect	Control		0.5% continuously		0.1% continuously		0.5% days 6-15 postcoitum		0.1% days 6-15 postcoitum
	13 d	21 d	13 d	21 d	13 d	21 d	13 d	21 d	13 d	21 d
No. pregant	9	10	10	8*	9	9	9*	8*	9	7
% pregnant	90.0	100.0	100.0	88.9	90.0	90.0	100.0	88.9	90.0	70.0
No. total resorptions	1	2	1	0	1	0	1	0	1	0
Av. No. corpora lutea	-	15.3	-	14.0	-	14.3	-	12.8	-	15.5
Av. No. implantations	13.0	13.4	14.3	14.4	12.8	13.6	16.3	13.4	13.3	14.4
Av. No. resorptions	2.0	3.9**	0.3	0.0	1.3	0.0	1.8	0.0	0.6	0.0
Av. No. live fetuses	-	11.6	-	14.4	-	14.3	-	13.4	-	14.4
No. dead fetuses	-	0	-	0	-	0	-	1	-	0
No. fetuses abnormalities	-	0	-	0	-	1	-	2	-	0
Av. weight fetuses (g)	-	3.5	-	3.5	-	3.9	-	3.5	-	3.7

 

* Deaths earlier had reduced these groups in number

** Significantly different from other females sacrificed at 21 d (p<0.05)

 

 

TABLE 3: EFFECTS ON THE REPRODUCTION OF SECOND GENERATION PARENTS (F1b) AND ON THEIR FIRST LITTERS (F2a)

 

	Control	0.5% continuously	0.1% continuously	0.5% days 6-15 postcoitum	0.1% days 6-15 postcoitum
No. pregant	19	15	20	19	18
% pregnant	95.0	75.0	100.0	95.0	90.0
Av. No. born live per litter	10.2	9.1	8.9	9.1	10.3
Av. pup weight at 4d (g)	9.7	9.5	10.4	10.1	9.6
Av. No. weaned per litter*	7.1	7.4	6.7	7.0	7.4
Av. weight of pups  at weaning (g)	43.0	41.7	46.0	45.5	42.9

 

* Litters containing more than 8 pups were reduced to that number at 4 d

 

 

 

TABLE 4: EFFECTS ON THE REPRODUCTION OF SECOND GENERATION PARENTS (F1b) AND ON THEIR SECOND LITTERS (F2b)

 

Effect	Control		0.5% continuously		0.1% continuously		0.5% days 6-15 postcoitum		0.1% days 6-15 postcoitum
	13 d	21 d	13 d	21 d	13 d	21 d	13 d	21 d	13 d	21 d
No. pregant	10	10	10	7	10	9	9	9	10	10
% pregnant	100.0	100.0	100.0	70.0	100.0	90.0	90.0	90.0	100.0	100.0
No. total resorptions	0	1	1	0	1	0	2	1	1	0
Av. No. corpora lutea	13.9	14.2	13.6	11.9*	13.6	13.6	13.4	13.4	13.2	13.8
Av. No. implantations	14.3	13.1	12.0	10.7**	13.0	13.4	13.3	11.2	13.0	13.5
Av. No. resorptions	1.2	1.4	0.9	0.9	1.8	0.9	1.9	3.8	1.3	0.9
Av. No. live fetuses	-	13.0	-	9.9	-	12.4	-	9.1***	-	12.6
No. dead fetuses	-	0	-	0	-	0	-	0	-	0
No. fetuses abnormalities	-	5	-	1	-	1	-	0	-	3
Av. weight fetuses (g)	-	4.8	-	5.4	-	5.4	-	4.9	-	5.2

 

*Significantly less than control rats at 21 d (p<0.05)

** Significantly less than rats fed 0.1% days 6-15 postcoitum (p<0.05)

*** Significantly less than control rats (p<0.05)

 

 

TABLE 5: INCIDENCES AND DISTRIBUTION OF FETAL ABNORMALITIES OBSERVED IN RATS

 

	Control		0.5% continuously		0.1% continuously		0.5% days 6-15 postcoitum		0.1% days 6-15 postcoitum
	F1c	F2b	F1c	F2b	F1c	F2b	F1c	F2b	F1c	F2b
No. litters	7	9	8	7	9	9	7	8	7	10
No. fetuses examined	93	118	115	69	122	113	94	74	102	128
No. fetuses examined for skeletal defects	31	37	38	22	39	38	30	24	33	43
No. fetuses examined for soft-tissue defects	62	81	77	47	83	75	64	50	69	85
Fetuses with gross abnormalities (%)	0	2.5*	0	0	0	0	1.1	0	0	0
Fetuses with skeletal defects (%)	0	0	0	0	2.5~	0	3.3	0	0	0
Fetuses with soft-tissue defects (%)	0	5.0#	0	2.1	0	1.3	1.1	0	0	3.5
Facial dysgenesis	-	1.7	-	-	-	-	-	-	-	-
Hematoma	-	0.8	-	-	-	-	-	-	-	-
Hydronephrosis	-	2.5	-	-	-	1.3	-	-	-	1.2
Double aorta	-	1.2	-	-	-	-	-	-	-	-
Cryptorchism	-	-	-	2.1	-	-	-	-	-	-
Hydro-encephalocele	-	-	-	-	-	-	1.1	-	-	-
Ascites	-	-	-	-	-	-	-	-	-	1.2
Hyperplastic testicle	-	1.2	-	-	-	-	-	-	-	-
Abdominal hemorrhaging	-	-	-	-	-	-	-	-	-	1.2
Missing rib, L 12th	-	-	-	-	2.5	-	-	-	-	-
Sternoschisis	-	-	-	-	-	-	1.1	-	-	-

 

* Percentages are based upon total number of fetuses examined

~ Percentages are based upon number cleared and stained

# Percentages are based upon number of animals examined for soft-tissue effects

Conclusions:

In a two-generation feeding study (reliability score 2), conducted to a protocol similar to OECD Test Guideline 416 and pre-GLP, HEDP (2-3Na) was administered to rats in doses of 0, 0.1 or 0.5 % (equivalent to 0 mg/kg bw/day, approximately 112 mg/kg bw/day and approximately 447 mg/kg bw/day respectively), either continuously to both sexes and to females only on Gestation Day 6-15. A significantly increased number of stillborn occurred in the highest dose groups of females dosed on Gestation Day 6-15). Additionally, the average number of corpora lutea was significantly decreased in the highest dose group in females dosed continuously for 21 days (although not 13 days) compared to the control group. There was also a significantly reduced average number of live foetuses compared to the control group although this was only observed in the F2b generation in the highest dose group in females dosed on Gestation Day 6-15, however, not in continuously dosed dams. In conclusion, no treatment-related malformations or developmental variations were noted at any exposure level. The occurrence of fetotoxic effects cannot be excluded at high doses which surpass the general systemic NOAEL in rats by a factor of 6 and are therefore likely to induce maternal toxicity. A NOAEL for maternal animals and foetuses was concluded to be 447 mg/kg bw/day (equivalent to 368 mg/kg bw/day active acid).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Reliability score 2

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In the key two-generation feeding study (reliability score 2), conducted to a protocol similar to OECD Test Guideline 416 and pre-GLP, HEDP (2-3Na) was administered to rats in doses of 0, 0.1 or 0.5 % (equivalent to 0 mg/kg bw/day, approximately 112 mg/kg bw/day and approximately 447 mg/kg bw/day respectively), either continuously to both sexes and to females only on Gestation Day 6-15. A significantly increased number of stillborn occurred in the highest dose groups of females dosed on Gestation Day 6-15). Additionally, the average number of corpora lutea was significantly decreased in the highest dose group in females dosed continuously for 21 days (although not 13 days) compared to the control group. There was also a significantly reduced average number of live foetuses compared to the control group although this was only observed in the F2b generation in the highest dose group in females dosed on Gestation Day 6-15, however, not in continuously dosed dams. In conclusion, no treatment-related malformations or developmental variations were noted at any exposure level. NOAELs for maternal animals and foetuses were concluded to be 447 mg/kg bw/day (equivalent to 368 mg/kg bw/day active acid) (Procter & Gamble, 1971a).

In the key developmental toxicity study (reliability score 2), conducted according to OECD Test Guideline 414 and pre-GLP, HEDP (2 -3Na) was administered to in concentrations of 0, 25, 50 or 100 mg/kg bw/day to 20 New Zealand rabbits. In the pre-study, mortality was observed in dams administered 500 mg/kg bw/day after day 4 or 5. Therefore, the concentrations were reduced to 100 mg/kg bw/day in the main study which did not cause any toxicity to the dams or to the foetuses. It was concluded a maternal and developmental NOAEL is ≥ 100 mg/kg bw/day for rabbits (Procter & Gamble, 1971a).

In a limited study (reliability score 4), conducted according to a protocol for FDA guidelines for reproduction studies for safety evaluation of drugs for human use and pre-GLP, HEDP-H was administered to 20 female Long-Evans rats. The test concentrations included 0, 16.5, 110 or 330 mg/kg bw/day and was provided by oral gavage twice daily in a dose half the total daily dose during Gestation Days 6-15. HEDP-H did not cause any adverse effects at any dose levels and therefore, a NOAEL of >330 mg/kg bw/day was concluded (Monsanto, 1966).

In a non-standard dietary teratology study only available as a brief summary (reliability score 4), conducted prior to the adaptation to OECD Test Guidelines and pre-GLP, HEDP-H was administered to rats by oral feed. No adverse effects were observed and therefore, a NOAEL was concluded to be at least 0.5% (P&G, 1967).

See read-across justification in IUCLID Section 13.

Justification for classification or non-classification

Based on the available read-across data, no classification is required for the reproductive and developmental toxicity of HEDP-xK according to Regulation (EC) No 1272/2008.